0. Preamble
X-ray data is the only structural experimental data you collect on your protein/nucleic acid. All that hard work you've just put
into making cute constructs and elaborate co-expression schemes is worthless unless you collect good data from the crystals
you have grown.
X-ray data collection often seems more theoretically challenging that it actually is - but there are several important choices to
make and some knowledge of crystal symmetry is helpful. Theory is important to the extent that it is good to understand the
basis of what you are collecting, but the finer nuances of diffraction space are less important than making sure you've got the
parameters right in Denzo.
The unit cell is usually not the smallest unique volume in the crystal - that would be the asymmetric unit. Unit cells contain
from one to many asymmetric units, arranged in patterns characteristic of what symmetry is in the crystal (i.e. the space
group). Each asymmetric unit contains the same environment as any other asymmetric unit i.e. they are all equivalent to each
other. One asymmetric unit can be mapped to any other one by a combination of symmetry operations, and therefore an
entire unit cell (and hence crystal) can be built up from the contents from a single asymmetric unit and the symmetry
operators.
There is an inverse relationship between the dimensions in real space and the dimensions in reciprocal space (i.e. diffraction
space). The real space unit cell dimensions a,b,c,,, have corresponding reciprocal space counterparts called
a*,b*,c*,*,*,*. The relationships are:
a* is parallel to b
b* is parallel to c
c* is parallel to a
a is parallel to b*
b is parallel to c*
c is parallel to a*
X
X
X
X
X
X
0.2 Symmetry
Chapter 3 of Stout and Jensen provides a pretty decent introduction of symmetry in the crystalline environment, and I will
only quickly review it here. It takes some time to get used to the various interactions of symmetry however it does pay to
spend at least a little time trying to understand what is going on in your particular crystal form.
Symmetry in a crystal is constrained by the fact that one must be pack the identical asymmetric units into a unit cell, such that
the environment of each asymmetric unit is identical. These constraints reduce the number of possible types of symmetry to
relatively few:
Pure rotation axes (1-, 2-, 3-, 4-, 6-folds with no translational component)
Screw axes (rotation axes with a translational component down the axis)
Mirror planes
Glide planes (mirror planes with a translational component parallel to the plane)
Inversion centers
Mirror planes and inversion center symmetries inevitably invert the chirality of chiral centers (e.g. flipping L-amino acids to
D-amino acids), so are not compatible with compounds that aren't a racemic mixture of chiral molecules. Proteins and nucleic
acids are non-racemic mixtures of very chiral molecules, so the only symmetries that can occur in macromolecular crystals are
pure rotations, screw axes and pure translations. This simplifies things a lot.
Symmetry within the unit cell also imposes some limitations on what values the unit cell dimensions may occupy. The socalled seven crystal systems can be sorted according to what symmetries they must minimally contain:
Crystal System
Rotational symmetry
Triclinic
Monoclinic
Orthorhombic
Tetragonal
Trigonal
Hexagonal
Cubic
1-fold
none
2-fold
==90 degrees
three perpendicular 2-folds ===90 degrees
4-fold
a=b, ===90 degrees
3-fold
a=b, ===120 degrees
6-fold
a=b, ===120 degrees
3 and 2-folds
a=b=c, ===90 degrees
although in some cases the crystals will contain more than the minimum symmetry. In turn the systems can contain one or
more Bravais Lattices:
Crystal System Lattice
Triclinic
P
Monoclinic
P, C
Orthorhombic P, I, F
Tetragonal
P, I
Trigonal
P, R
Hexagonal
P
Cubic
P, F, I
These 14 Bravais Lattices provide no additional rotational symmetry, but may give additional translational symmetry to the
unit cell. P is Primitive (no additional symmetry), C is C-face centered: for each atom at (x,y,z) there is another one at (x+1/2,
y+1/2, z) where the "1/2" means "half of the unit cell edge along that direction". I is body-centered, so that for each atom at
(x,y,z) there is another one at (x+1/2, y+1/2, z+1/2) and F is all-face centered, namely for (x,y,z) there are also atoms at
(x+1/2, y+1/2, z), (x+1/2, y, z+1/2) and (x, y+1/2, z+1/2).
Symmetry operators like the ones above are expressed in fractional coordinates where each unit cell location is expressed as
a linear sum of fractional unit cell translations:
XYZ = x.a + y.b + z.c
(where a,b,c are the unit cell vectors and x,y,z are the fractional scalars denoting a location within the unit cell). For
orthorhombic, tetragonal and cubic space groups the fractional locations (x,y,z) are the same as the more familiar cartesian
locations (X,Y,Z) divided by the unit cell edges i.e.:
(x,y,z) = (X/|a|, Y/|b|, z/|c|)
Fractional coordinates are periodic so you can add and subtract integers from them and it refers to the identical location (but in
adjacent unit cells). You can always map them to the range 0...1 by this addition or subtraction.
If your asymmetric unit contains more than one molecule, then you will also have non-crystallographic symmetry which has
some of the same constraints as crystallographic symmetry (no mirror or inversion symmetries) but can otherwise be in an
arbitrary position, direction and rotation. Except in very special cases (e.g. high-symmetry viral capsids), non-crystallographic
symmetry is not considered for the purposes of data collection but it can be very useful during map interpretation (averaging)
and refinement (ncs restraints). The downside is that it tends to make your unit cell bigger than it might otherwise be. As of
writing my "personal best" is 56 distinct momoners in the asymmetric unit with the larger crystal form of the 20S proteasome.
Symmetry in real space is a combination of rotations and translations that give rise to a unique pattern of symmetry elements
called the space group. Each space group is a member of a Bravais Lattice, and a Crystal Class. However in diffraction space,
the translational components of symmetries are not relevant to the symmetry of the diffraction pattern. Only the rotational
parts of the operator cause symmetry in diffraction space. As an example the symmetry operator (-x,y,-z) as found in the space
group P2 has the same effect in diffraction space as the symmetry operator (-x,y+1/2,-z) as found in the space group P21. It
generates symmetry between reflections (h,k,l) and (-h,k,-l) in both cases. This means that several different space groups can
have the same diffraction symmetry, because the relative location of symmetries is not relevant (only their direction and type).
P2 and P21 have exactly the same diffraction pattery symmetries for this reason. Different space groups that have the same
angular relationships of symmetry elements are said to have the same point group.
For example, the space groups in the monoclinic crystal system must have a single 2-fold rotation or screw axis along the baxis of the unit cell (by convention). There are three of these space groups for proteins and nucleic acids: P2, P21, C2. Both
P2, P21 are Primitive lattices and so can be called Primitive Monoclinic, but C2 is C-face centered Monoclinic. Since these
are different space groups the precise arrangement of the symmetry elements is different between each space group, i.e. the
symmetry operators in different locations in real space. However they all have a 2-fold axis parallel to the unit cell b-axis.
When translation components of the symmetries are removed, they all have the same symmetry in diffraction space, i.e. that of
point group 2, or actually 2/m if you take into account Friedel's Law. For P2, P21 and C2 the intensity of reflection (h,k,l) is
identical to that of reflection (-h,k,-l). If one includes Friedel's Law (h,k,l) is also related to (-h,-k,-l) - note the inversion
symmetry here - and (h,-k,l). Freidel's Law applies for native datasets but the extra symmetry is broken in the presence of
significant anomalous scattering.
Crystal System
Triclinic
Point Group
1
Laue Class
-1
Monoclinic
2/m
Orthorhombic
Tetragonal
222
4
422
3
32 (312 and 321)
6
622
23
432
mmm
4/m
4/mmm
-3
-3m
6/m
6/mmm
m-3
m-3m
Trigonal
Hexagonal
Cubic
Note that because crystal classes specify only certain minimal symmetries, there is often more than one point group per crystal
system. For simplicity I only show the protein-relevant point groups here (there are others).
It can be shown that in the absence of anomalous scattering, that the intensity of the reflection with Miller indices (h,k,l) is the
same as that of the reflection (-h,-k,-l). This is called Friedel's Law. The consequence of Friedel's Law is that even if the
space group lacks a center of symmetry, the diffraction pattern is centrosymmetric. In this case, point group 2 becomes point
group 2/m by the action of Friedel's Law, and point group 222 becomes point group mmm, etc. mmm is called the Laue Class
of the point group 222. While I don't suggest memorizing Laue classes, you should be aware of their existence and the extra
symmetry that gives rise to them. For SAD, SIRAS or MAD data collection, which relies on anomalous scattering, Friedel's
Law is invalid. Friedel's Law does not apply where anomalous scattering is significant. We generally ignore the very small
amount of anomalous scattering that occurs from light elements (C, N, O, S, P) at typical wavelengths used in data collection.
It's there, but it's way down in the noise level.
During data collection, the various symmetry-related reflections are observed independently. During data integration
(DENZO), these reflections are also processed independently. However during data scaling (SCALEPACK) these individual
observations of symmetry related copies of the "same" reflection are merged together to produce the unique data.
Unique data no longer has any symmetry redundancy within it - i.e. no reflection is related to any other one within the unique
set by crystallographic symmetry. Comparison of symmetry-related reflections that should be identical is the basis for most of
the data processing statistics, e.g. Rsymm . There's some ambiguity over the usage of Rsymm and Rmerge - I used the former
(symm) to refer to internal agreement with symmetry, and I use the latter to refer to the merging R-factor when I merge
multiple datasets together. Not everyone follows this rule. The PDB asks for both Rmerge and Rsymm upon structure deposition
but they seem hopelessly confused about what the difference is (or even if there is one).
poorly, can be more than 2 degrees. Freezing crystals often causes their mosaicity to increase because of the dynamics of the
freezing process. You can collect data from crystals with high mosaic spreads, but generally the data is not quite as good as
that from crystals with low mosaic spreads because the scattering density is spread out over more pixels.
The scattering (diffraction) due to a crystal whose unit cell contains electron density &rho(xyz); at any given point is given by:
which is a class of function called a Fourier Transform. The inverse of this equation depends on the amplitude (magnitude)
and phase of the structure factor (F) for each reflection (hkl).
The whole purpose of data collection is to measure the structure factor magnitude which as much accuracy as possible.
Currently there is no technology available which can measure the phase of the structure factor, which gives rise to the socalled "phase problem" in crystallography where we must deduce the phase by other means.
major source of systematic error in MIR, and the dominant reason the MAD technique was developed.
A first approximation for a standard stabilizing solution is about 1.05-1.2x the precipitant concentration in the well, with the
buffer, salt, additives etc kept the same. Remember to include contributions from the protein buffer/salt combination as well.
The idea is to use higher precipitant to compensate for the lack of protein in the stabilization solution, but to keep the other
components the same.
Since protein crystals are usually at least 50% water by volume, it follows that they are very sensitive to dehydration. If you
remove a crystal from solution it will dry out and disorder very quickly. Sometimes however as a check it is useful to
mount crystals without freezing them. To do this we must keep them in an environment saturated in water vapor to eliminate
evaporation. One possible way to do this is to mount the crystal after it has been immersed in oil - this is a technique
sometimes used when freezing crystals but more rarely used for "room temperature" mounting. The conventional way for nonfrozen mounting is using thin-walled glass capillaries. In this scenario a crystal is inserted into the capillary and the
surrounding solution slowly removed by pipette or some absorbing medium (filter paper, paper wicks etc). The crystal is never
completely dried out, and adheres to the side of the capillary tube via surface tension. The tube is sealed with wax or oil plugs
and the crystal is maintained in an environment that is saturated in water vapor but is otherwise somewhat similar to being in
solution. There are enough minor technical issues with capillary mounting that a full description of my mounting technique
alone would take too long for the purposes of this course.
The major downside of mounting at room temperature (or 4 deg C) is that the crystals experience rapid radiation damage.
Cooling the crystals to 4 deg C helps a little, although not all crystals tolerate the transition. Some much lower temperatures
have been achieved in the interests of studying enzyme mechanisms but the apparatus for doing those sorts of experiments is
cumbersome. Radiation damage comprises two components: a dose-dependent component due to ionization of protein and
solution by X-ray photos; a time-dependent component due to generation of free-radicals and the propagation of ongoing freeradical reactions throughout the crystal. It's been shown that the time-dependent component of radiation damage is the
dominant one, and some crystals last as little as 10 hours on a home source (that would be about one minute's worth of X-ray
exposure at X25).
The routine method to substantially reduce radiation damage is to flash-cool the crystal in liquid nitrogen, liquid propane,
liquid freon or a cold (100K) nitrogen gas stream under conditions in which the solution "glasses". Glassing means that the
solvent molecules do not have crystalline order (i.e. glass vs ice), but the crystalline order of your protein crystal is
maintained. Normal water and dilute buffers form micro crystalline solid phases when frozen like this - they appear opaque,
but the addition of cryo-protectants like glycerol can make the solid phase become amorphous, resembling a glass. Freeradicals still form with X-ray exposure but are locked in place in the frozen crystal and this prevents their propagation around
the crystal, and basically stops their reactivity. Dose-dependent radiation damage still occurs to those crystals, but timedependent radiation damage is largely halted. This turns out to reduce the radation damage rates of protein crystals by orders
of magnitude. Current research is underway to figure out if some additives can reduce the radiation damage even more by
"mopping up" some of the dose-dependent damage too.
Adding glycerol to a final concentration of 30% (v/v) is often a fairly efficient way to generate a cryo-capable solution from
most crystallization conditions. Glycerol is by far the most frequently-used cryoprotectant. In fact some crystals can even be
induced to grow in enough glycerol to be a cryoprotectant without adding more - this simplifies handling and reduces the
change in environment that a crystal must experience. Hampton Research make a version of Crystal Screen called Crystal
Screen Cryo which is the standard Crystal Screen condition with enough glycerol added to make them all cryo buffers. This
will give you an idea of how much glycerol is required for various conditions - generally salts need ~30% except near
saturation, PEGs need 15-30% depending on their concentration since they act as partial cryoprotectants themselves. Alcohols
sometimes need closer to 35% glycerol.
I advocate the use of rapid stepwise equilibration in changing the environment of a crystal from 0% glycerol to 30% glycerol.
I do it in 10% v/v glycerol increments. You can also dunk the crystal in the final concentration of cryoprotectant and mount
immediately, but I personally suspect that the stepwise method tends to work better on average. The downside of the stepwise
is that the crystal gets moved from solution to solution more often. The downside of the "dunk and go" method is that the
change in environment from 0 to 30% glycerol is really quite abrupt.
Although glycerol is the most popular, many other cryoprotectants can be used: ethylene glycol, xylitol, sucrose, PEG-400,
MPD have all been used fairly frequently in data collection at liquid nitrogen temperatures. Start off with glycerol and then
check one or more other cryoprotectants to make sure that glycerol is not hurting your diffraction properties. There is also an
online database of cryo conditions by JAXA.
You should never assume that your handling of the crystal is inevitably benign, especially if your diffraction properties
are fairly poor. There are many examples of crystals that are extremely sensitive to environment or don't like one
cryoprotectant solution or another. Until you have "good enough" diffraction you should at least explore alternatives. One
possibility is crystal annealing which has sometimes been found to radically improve diffraction from crystals. One can do
this in situ by blocking the N2 flow onto the crystal (a piece of paper or piece of thin flat plastic works well), allowing it to
thaw (30 sec or more), then restoring the flow to re-freeze it. You can also remove the crystal, let it thaw, put it back in cryo
solution and then remount it. In any even if you have bad diffraction and the crystal is unusable there's really no point not
trying this method.
These two images were taken from http://srs.dl.ac.uk/OTHER/NEWSROUND/Issue_10/px10.htm to illustrate the potential
power of crystal annealing:
Before
After
The simple set up involves a magnetic cap (attaches to goniometer head on the X-ray machine), a thin metal pin and a thin
fiber loop of variable size (0.05-1.0 mm, typically). Pins are glued into the bases with epoxy (Hampton sells mounted
cryoloops). The caps fit on fairly standard magnetic bases (see which attach to or are integral parts of existing goniometer
heads. Hampton's Crystal Cap HT is my current favorite since this works especially well with the NSLS X29 and X25
beamline goniostats. Previously the standard Crystal Cap and Crystal Cap Copper were my standards. The "copper" version
helps to reduce icing on home sources because of the greater heat transmission by the copper sleeve but it's not compatible
with the cryo tongs so routine application is somewhat limited. Icing on the pin is a relatively rare problem at synchrotron
beamlines due to the short duration of data collection. The associated cryo vial serves as a reservoir for liquid nitrogen when
handling and transporting frozen crystals. Vials attach to the bases either via screw mounts or via magnets. Most of the robots
for auto-mounting at synchrotrons have converged on a standard of Hampton mounts (usually the low profile all-metal HT
mounts, shown center in the figure above) with a base to loop distance of approximately 21mm (e.g. see the X6a Wiki for the
automounter). The loops are made of 20 or 10 nylon thread - typically we prefer the 20 kind which seem to be less likely
to move ("wave in the breeze") in the cryostream.
The early 1990's saw gradual acceptance of the method as generally applicable, including dispelling concerns that it would
distort the protein structure or introduce excessive non-isomorphism. Many crystal structures e.g. the CDK2:CyclinA structure
from the early days of the Pavletich lab would have been completely impossible without it as the crystals died overnight on
the comparatively weak home source at 4 deg C. During that era cryo crystallography became the standard method for
macromolecular data collection.
Cryo "wands" plug into the bottom of the caps. For the
standard CrystalCap (incl the Coppers) the bases have a
locator tab that allows the wand to fix in place and unscrew
the cap from the threaded vial. The magnetic HT bases don't
have tabs, so the wand has an internal plunger mechanism to
displace the cap from the wand. A close-up of the CrystalCap
in place on the wand (with the tab located) is shown in the
second picture.
Mount an unused loop of the same standard dimensions and center it to get the right spindle height etc
Optionally back off the nozzle of the cryo system a little to give you a little more working space
Fill a shallow and wide dewar with fresh liquid nitrogen
Take the cryo tongs (dry them as necessary) and put them in the lN2 to cool
Take the magnetic wand (metal rod with magnet and locator tab at the end) and cool the end of it
In the following steps make sure that the crystal remains under the lN2 until the last transfer step.
Remove a pre-frozen crystal from a cane, and immerse it in the lN2 in the shallow dewar (forceps help with this)
Locate the magnetic end of the crystal loop base, and plug the manetic wand into that end of the cap
Make sure the locator tab on the wand mates with the slot on the base of the cap
Unscrew the loop from the cryo vial - the crystal is now open in the lN2 so do not bang it into anything
Assuming the tongs are completely cooled to lN2 temperatures, open the tongs and move them around the crystal
Close the tongs around the crystal, make sure they are fully closed and enclose the crystal
Remove the magnetic wand from the loop base - the crystal is now held by the tongs only
Remove the tongs from the lN2 and rapidly but smoothly transfer the crystal to the goniostat
The thermal mass of the tongs ensures that the crystal does not thaw, and temperature is maintained once the tongs are
within the cryo nitrogen gas flow
Once the crystal base is firmly seated on the goniostat, open the tongs and put them to one side
Inspect the crystal to make sure it hasn't thawed, then center it and proceed as normal
Similarly the protocol for removing a crystal from (e.g.) a home source for relocation to a synchrotron is:
Fill the shallow wide dewar with fresh lN2
Cool the cryotongs thoroughly in the lN2
Rapidly remove the tongs from the lN2 and clamp around the crystal still on the goniostat
Make sure that the tongs surround the crystal, then remove from goniostat and return to lN2
Cool magnetic wand in lN2, then plug it into the crystal cap base, with the locator tab in the correct orientation
Once the cap is firmly on the wand, open the tongs and remove them
Take a crystal cap on a pair of forceps, and cool it thoroughly in the lN2
Move the cap to cover the crystal and screw the crystal down tight into the cap
Use forceps or your gloved hand to place the crystal plus vial full of lN2 onto a cryo cane and store in a large dewar
For obvious reasons, fresh lN2 with no ice is important for both steps, as is dry apparatus (ice will form on tongs left to warm
up in air). Ethanol is a pretty good way of displacing ice. A certain amount of practice is necessary to get the manual
manipulation aspects working fast enough without inadvertently removing the crystal from the lN2 (which guarantees disaster
via icing and rapid thawing).
At X29, X25 and probably some other beamlines their cunning design of their goniostat enables you to mount pre-frozen
crystals straight from lN2-filled vials right onto the goniometer head. This has saved us a LOT of time at these beamlines.
bands "boil off" the filament, accelerate down the tube under the
potential difference, and smack into the target. When they do so,
they ionize electrons from the target material. When these (or
other) electrons drop back into these vacated energy levels, they
give off energy partially in the form of electromagnetic radiation.
Plus a lot of heat. If the electrons are ejected from the lowestenergy orbitals (1s, 2s etc) then a lot of energy is released when
electrons reoccupy them - released as X-rays. Metal targets are
used because these do not damage much with electron
bombardment and conduct heat efficiently (most of the energy is
lost as heat, not X-rays). Beryllium windows, relatively
transparent to X-rays, let the X-rays escape the evacuated tube.
Rotating anodes: these are the same idea as a sealed tube, except
the anode is spinning (e.g at 6,000 rpm), allowing a much greater
loading to be put on the target since the heat is spread out over a
larger area. Other than that the differences are purely engineering:
a 2 lb copper target spinning at 6,000 rpm places some stress on
bearings and seals; the vacuum system is no longer a single
assembly and typically has two different pumps; wear and tear is
significant and maintenance becomes a more serious issue. The
state of the art for macromolecular rotating anode sources is the
Rigaku/MSC FR-E generator which is a lower power but high
brilliance X-ray generator.
In both sealed tube and rotating anode sources the wavelength is
fixed by the characteristic emission spectra of the target
material. Copper is the one most often used for proteins since it is
hard, an efficient conductor of heat, and the CuK emission is
relatively intense. The wavelength of the X-rays produced is 1.54
. Small molecule crystallographers typically use weaker
Molybdenum sources, with a wavelength closer to 0.7 , since the
higher-energy X-rays are absorbed less by the experimental
mount, etc.
The primary synchrotrons within the USA are: the National Synchrotron Light Source (NSLS) at Brookhaven National Lab;
the Advanced Photon Source (APS) at Argonne National Lab; the Cornell High Energy Synchrotron Source (CHESS) at
Cornell College; Advanced Light Source (ALS) in Berkeley; Stanford Synchrotron Radiation Lab (SSRL) in Stanford
California. Other notable ones include Diamond (UK), ESRF (France), Photon Factory (Japan) etc - consult synchrotrons of
the world for a fuller list.
Bending magnet beamlines at Brookhaven (NSLS) are 50-100x brighter than a home source (e.g. X12C, X9A). Wiggler
beamlines at Brookhaven (e.g. X25) are about 10x brighter than bending magnet beamlines. At Argonne (APS), the bending
magnet beamlines are at least 1000x brighter than a home source and the undulator beamlines are 10-100x brighter than the
bending magnets. The actual brightness, beam shape, wavelength tunability and other factors are all heavily dependent on the
design of the beamline optics as well as the synchrotron itself. Nearly all modern beamlines have optics with energy resolution
and energy tunability properties suitable for MAD data collection (CHESS A1 and F1 beamlines are notable exceptions).
since there is no mechanism to get rid of the "white radiation" background and the CuK is only partially absorbed. Mirrors
are used at synchrotrons for beam focussing only, with wavelength selection achieved using separate monochromators.
Mirrors on home sources usually have better-defined beams (smaller) than the corresponding monochromator optic.
Multilayer Optics: The most recent advances along the lines of mirror optics have involved multilayer coated mirrors.
Precisely controlled coatings of the mirrors with specific layer spacings show a distinction of having a high efficiency of
reflection at specific angles with a narrow band-pass in wavelength, so that not only are the multilayer mirrors more efficient,
they also enhance spectral purity. These are the state of the art with in-house systems which exhibit fixed-wavelength
installations. However the narrow range of wavelength applicability because of the defined layer spacing makes them
unsuitable for synchrotrons which usually require optics that work over a wide range of wavelengths.
Collimators: these are just pinhole devices to reduce X-rays, which might otherwise propagate in all three dimensions, into a
thin controlled beam. Typical collimators are just metal tubes with two aligned pin-holes at each end. The pin-holes usually
have diameters in the range 0.1-0.3 mm. Collimators do not change the spectral purity of the X-rays they are just a physical
device to limit the beam by basic geometry. On mirror systems they mainly serve as a device to limit air-scatter by the beam
from reaching the detector. On a synchrotron they do tend to clip the edges of the beam and we tend to see better results from
smaller (0.1) collimators than larger ones.
Vendor websites
Area Detector Systems Corp (ADSC)
MAR Research Inc
Rigaku-MSC
Properties of ideal X-ray Detectors :
High efficiency - all x-ray photons converted to signal
Detector Quantum Efficiency (DQE) = (S/N output) / (S/N input)
A very good detector has DQE ~0.8
Stable with respect to time, temperature, environment.
No geometric distortion
Scaleable with count rate
Uniformity - every pixel has the same response
High counting rates (synchrotrons provide >100,000 counts/second)
High dynamic range (ratio of strongest:weakest signal of 105:1 or 106:1)
Large active area
High spatial resolution (film 50-25 microns; image detectors 100-200 microns)
Fast readout
Compact and light (to move or incline relative to the sample)
Geiger counters: everyone knows that Geiger counters can count X-rays via ionisation events. They can do it pretty well,
within certain limits. In fact older diffractometers counted one reflection at a time using this ionisation chamber technology,
and if your crystal lasted long enough one got very good data indeed. Collecting a 250,000 reflection dataset one reflection at a
time will take you a while, which is the whole reason area detectors were invented.
Multiwire proportional counters: these extended the ionisation chamber idea - these were some of the first true "area"
detectors, and were part of the wave that revolutionized protein data collection in the 1980's. The Xuong-Hamlin (UCSD)
model was the first one that was used for routine data collection. These detectors contain a 2D grid of wires in a medium that
was ionised by X-rays. The ionisation events are detected as electronic signals on pairs of wires in the x and y directions,
producing a 2D electronic image of diffraction. The most popular of these detectors was the Xentronics/Nicolet/Siemens
detector, still in use in some labs today, and also the older Xuong-Hamlin UCSD design detectors (the original ADSC
detector). Multiwire detectors cannot deal with high flux, however (their ionisation medium saturates, as does the detection
circuitry), so they were not effective a synchrotron sources, and even for well-diffracting crystals on home sources (e.g.
lysozyme).
Ionisation type detectors literally count photons in a serial manner - so-called photon counting detectors. The remaining
detector technologies "integrate" the signal by accumulating the counts to be read out later.
Film: if you've ever left high speed photographic film in checked-in luggage you'll realise that X-rays fog film. Film was the
first X-ray recording medium (Roentgen ~1895), and was commonly used at places like synchrotrons up to about 15 years
ago. Film suffers from a limited dynamic range, a fair amount of background noise (chemical fog) and principally that it's a
pain to develop and scan all those images. Film generally has higher spatial resolution than most image plate detectors but
newer CCD detectors get close. Most modern CCD and image plate detectors usually offer more active area than the old film
packs did (12x12cm ?).
Image plate detectors: Image plates (storage phosphors) arose as popular alternatives to film in medical labs - X-ray photons
cause charge to accumulate in Europium-doped matrials that coat these flexible plates. The metastable charge can then be read
out by photostimulated luminescence with a laser. The image plates are then "bleached" with white light before re-use to
remove any remaining signal.
Image plates are larger than most other area detectors, fairly sensitive, have a large dynamic range. The R-AXIS and MAR
detectors came to dominate (and still dominate) home source data collection. The R-AXIS series of detectors utilize two
plates, so that one plate may be read while the other one is being exposed. The R-AXIS IIc has smaller rigid plates, while the
R-AXIS IV and IV++ have flexible plates mounted on a steel belt.
CCD detectors: CCDs are small light-sensitive computer chips that are used extensively in modern digital cameras (and spy
satellites). In X-ray detectors, the X-rays first strike a gadolynium oxysulfide phosphor screen at the front of the detector, the
phosphor image is reduced in size by a fibre-optic taper then projected onto the CCD chip. The taper is necessary in order to
increase the active area of the detector over the rather modest size of the CCD chip itself (most CCD chips are of the order of
1-5 cm). The very first version of this for routine use in crystallography was the FAST detector by Enraf-Nonius.
Good CCD chips (as opposed to the junk in most consumer digital cameras) are expensive to make, especially the larger ones,
so many CCD detectors comprise several small 1 or 2 megapixel (1K x 1K pixel = 1 megapixel) CCD chips stacked side-byside. The most popular one is the ADSC Quantum210 with four 1 megapixel chips in a 2x2 array. ADSC now also make a 3x3
array of 4 megapixel chips (2K x 2K), the Quantum315. The Quantum315 is on the X25 beamline at NSLS and on most
APS/Argonne beamlines, while the Quantum210 is on CHESS A1 and F1. CCDs are sensitive, but suffer from electronic
noise (they must be cooled to reduce this) and are sensitive to environmental radiation (the so-called "zingers") including
radiation originating in the fiber-optic taper. Their dynamic range is only moderate, a deficiency most often exposed at
synchrotron sources where low-resolution reflections can become saturated on longer exposures ("overloads").
Newer technologies: Crystallographers tend to use whatever technologies have been developed by others: multiwire photon
counters were developed by high-energy physicists, image plates for radiology, CCDs for spy satellites and digital cameras.
New technologies like Pixel Array Detectors (using the photoelectric effect) and Amorphous Silicon Detectors will probably
filter their way down as X-ray crystallography detectors once they become more widely available. MAR appear to be
developing detectors based on solid-state semiconductors that detect X-rays directly. GE are doing the same. So far these do
not seem to have penetrated the market and the image plate detectors still dominate home sources, as do the CCD detectors for
synchrotrons.
Why do we use CCD detectors at synchrotrons?: they are relatively sensitive area detectors with a resonable dynamic range,
fast readout time, and reasonable active area. Although they have a smaller area than a typical image plate (the Q315 is,
however, large), the much faster readout time (less than 10 seconds vs. ~2 minutes) is an enormous advantage at a
synchrotron, where exposure times are typically 5-40 seconds and the amount of dead time between exposures is a huge factor
in data collection efficiency, often exceeding exposure time at the newest synchrotron sources.
Why do we use image plate detectors in house?: Image plates are large, sensitive and have a large dynamic range. In fact their
only significant problem is that they take a relatively long time to read out (2-4 minutes with Raxis IV, IV++). This isn't a big
issue with in-house data collection where exposure times are 15-60 minutes an image. CCDs are less useful in-house: they are
more sensitive than image plates, but they also suffer with much more inherent noise than image plates for the same exposure
time (from both zingers and electronic noise). For weakly diffracting crystals on modest intensity sources, image plates tend to
give better data.
Your crystal is mounted onto a small precision goniometer head that allows
for precise adjustment of the translations (and in some cases limited rotation
via arcs) of the crystal. On most beamlines this is what you use to center the
crystal. On some newer beamlines there is now a separate centering
mechanism so you don't need to do that. Supper also offers goniometer heads
with detachable extended arcs for easier mounting of pre-frozen crystals as an
alternative to cryo-tongs.
The mechanism that the goniometer head attaches to is called the goniostat
and these are designed to allow precise positioning of the centered sample at a
wide array of positions. Some goniostats are simple 1-circle designs (like the
one on our area detectors) with a single (phi) axis. Other more elaborate
ones may consist of a large circle on which the rotation axis may be
positioned arbitrarily around a circle, as shown at left. These are the 3- and 4circle goniostats, of which the majority are made by the Huber company in
Germany. The various angles tend to be , , , 2. You most often see these
at synchrotrons since they are both expensive and large, although also
versatile. A more compact design that still allows much flexibility is the kappa
goniostat by Enraf-Nonius that has a specific inclination of the axis to the
axis to allow for a relatively large range of accessible crystal angles with
relatively small bulk. Normally the detector is arranged such that the direct
beam would strike the detector approximately at its center - the 2 axis on
many gioniostats allows the detector to be offset from the beam and is
especially useful when trying to collect high resolution data on small
detectors or large unit cells (when the detector has to be moved further back).
There's also the cryo unit. These come in two basic designs - those that use liquid nitrogen and those that use nitrogen gas as
the main source. The simplest design is simply source of dry nitrogen gas (via boil-off) that is cooled by passing it via a metal
coil through a liquid nitrogen dewar and is then blown at the crystal. The very first Rigaku/MSC systems were like that. The
Oxford cryostream system such as the one at Princeton uses a different method - it sips lN2 which it then heats to room
temperature gaseous form for flow control before cooling it back down (via heat exchange from the incoming lN2) to 100K
and blowing it at the crystal. Lastly the X-STREAM and X-STREAM 2000 systems from Rigaku/MSC purify their own N2
gas from the air, and then cool it via a helium refigeration pump. All systems use a laminar coaxial flow of room temperature
dry nitrogen surrounding the core of cold nitrogen gas to reduce sample icing via mixing with room air.
The single, critical, thing that Bragg's Law imparts is as follows: scattering from a crystal occurs in all directions. However
the scattering is only visible in a finite number of directions that obey the above law, i.e. the path difference between waves
scattered by adjacent lattice planes is a multiple of the wavelength of the radiation - the waves are in phase and constructively
interfere.
vectorial. The diffraction vector (S) is defined as being perpendicular to the planes that originate diffraction in Bragg's Law.
The length of the diffraction vector is the reciprocal of the spacing between the planes (1/d). In terms of the reciprocal space
unit cell vectors a*, b*, c*:
The reciprocal space unit cell axes have defined directions with respect to their real space counterparts (a, b, c). Namely, a* is
perpendicular to the plane containing b and c. (b* perpendicular to a/c; c* perpendicular to a/b). These are geometric
consequences of the Laue conditions.
There sphere has a radius of 1/. The crystal sits at the center of the sphere. In the diagram above the X-ray beam comes from
the left. The unscattered (direct) beam passes through the crystal and the point where it reaches the sphere surface is the origin
of reciprocal space. For a diffraction point in reciprocal space to be in diffraction condition, it must lie on the surface of the
Ewald sphere. The angle between the indident and diffracted beams is 2 and the vector connecting the reciprocal space
origin and the diffraction point is the diffraction vector.
Visualization of the Ewald sphere construction is useful in data collection because it gives a way to understand which points
are in diffraction condition. In the diagram below a "prefectly aligned crystal" is arranged such that the C* axis is pointing
down the C/C*-axis, and that the reciprocal lattice planes are pependicular to the beam. Lattice points are shown in gray, and
those in diffraction condition are shown in blue.
Even though the Ewald sphere is in reciprocal space (inverse distance) and detector geometry is in real space, we can use the
predicted angles of diffraction (2) to predict the diffraction pattern measured by a by a detector given a known instrument
geometry. We do this based on ray-tracing or similar triangles.
Since reciprocal space can be viewed as consisting of planes of reciprocal lattice points, the diffraction pattern appears as if it
is comprised of diffraction spots arranged on a series of ellipses. For the perfectly aligned crystal the rings are all circular
since the planes are perpendicular to the beam. Notice that the L=0 plane doesn't show up on the pattern in this situation
because only the (0,0,0) reflection is in diffraction condition and is buried underneath the beam stop. Note that (0,0,0) is
always in diffraction condition but we cannot measure it directly because it is swamped by the X-rays in the direct beam that
did not interact with the crystal.
As the crystal rotates, the reciprocal lattice rotates in the same way as the crystal and the planes become inclined to the direct
beam (which is usually the viewing angle). The projection of the circles onto the detector renders them as ellipses. Also notice
that since the planes are now inclined, more of the L=0 level is visible on the detector and that different parts of the L=1 and
L=2 planes are in diffraction condition.
The physics of oscillation images are relatively easy to understand, since all they involve is
rotating the crystal through a small solid angle about a single axis. The pattern you see
corresponds to planes in reciprocal (diffraction) space slicing through the Ewald sphere so
that only a limited amount of each lattice plane is in diffraction condition within the
oscillation range. Entire datasets are built up by collecting contiguous series of such images
to form a solid volume of rotation. This is the method that we use to collect all our data.
This is variously know as the "rotation method" or oscillation method. The ability to autoindex oscillation data has considerably enhanced the usability of this method.
Weissenberg
Precession
As the name suggests, precession photography involves making a crystal precess at a fixed
angle around a defined axis. If the crystal is precisely aligned such that a real space unit cell
axis lies along the rotation axis, a precession photograph can be arranged to provide view of
a single plane through diffraction space. Since this involves introducing a metal layer screen
that blocks most of the diffraction that is happening and only allows passage of that from the
desired layer, it is an incredibly inefficient way of collecting data. However it was used in
the early days of protein crystallography before advanced algorithms for auto-indexing
oscillation photographs made the interpretation of those more straightforward. I did a lot of
precession photography when I was screening for heavy atom derivatives as an
undergraduate (1986). The Pavletich lab bought a precession camera in 1993 but it was
never installed. That should tell you something. The method produces an undistorted view
of a single reciprocal lattice plane. In the (very) old days you used to compare zero-level
projections (0kl, h0l, hk0) between natives and potential heavy atom derivatives to look for
relative intensity changes. These days you can do the same thing in a fraction of the time
using conventional oscillation photography.
Laue (polychromatic)
In contrast to methods that have been discussed before, all of which use monochromatic Xrays, Laue photography specifically uses polychromatic X-rays over a wide wavelength
range. This is the same thing as if you made the Ewald sphere more like a solid ball than a
thin shell like a ping-pong ball. The advantage of Laue is that many, many diffraction
maxima are in diffraction condition at the same time, so we can collect the data in one or
just a few images. Laue data collection held promise in the early days, especially for highsymmetry space groups and time-resolved studies, but the inherent difficulties in indexing
the diffraction images from these systems, with multiple overlapped spots from multiple
wavelengths, has essentially rendered it useless for routine data collection. In fact very few
Resolution Limits
Although collecting 1.0 data on a project is a cute idea, the amount of useful biological information that you can extract
from a high resolution structure saturates at around 1.4 at which point the location of all well-ordered non-hydrogen atoms
should be well-defined. Most interesting biological structures don't diffract that far, so it's not normally an issue. But there is
limited value in going to ultra-high resolution unless you actually plan on studying the position of protons in your structure
(this might be relevant for enzyme mechanism, however).
Usually, what you're faced with is working at the low-resolution end. Experience suggests that you can get meaningful
biological data from structures at 3.5 resolution or better, but at worse than 4.0 you better either have another very similar
structure for comparison, or be working on something of epic importance (e.g. the ribosome). At 4.0 the conformation of
most side-chains will be questionable, and it will not be possible to trace your chain without ambiguity in many cases - the
biological information content of your structure starts to become pretty low.
Sometimes you can extend the resolution of your crystals by going to a brighter source. Very small but well-ordered crystals
may not diffract well in the lab because the beam is not very bright and it is quite large. Put these crystals in a synchrotron
beam and they often yield very good data at relatively high resolution. Conversely, large badly-ordered crystals will often
diffract as badly at the synchrotron as they do at home, because the strength of the X-ray beam is not limiting the resolution of
the data - the crystal order is.
The number of unique reflections in a dataset varies as the cube of the resolution - specifically, as 1/d3. This means there are 8
times more diffraction data points at 2 than at 4. Apart from the sheer advantage of increased optical resolution, having 8x
more points in your refinement alone will pretty much guarantee a much greater degree of accuracy in your structure.
centering operations in C, F and I lattices) gives the number of symmetry-related reflections in reciprocal space. However
Friedel's Law may double that number....
In the absence of significant anomalous scattering even the lowest symmetry space group P1 has two-fold redundancy in
complete data by Friedels Law:
I(h,k,l) = I(-h,-k,-l)
i.e. diffraction intensities show centrosymmetric symmetry even if your crystal does not (and protein crystals cannot have this
symmetry). Bear in mind that Friedel's Law is invalid in the case of anomalous scattering so you cannot use the above
relation while collecting SAD or MAD data.
Assuming that Friedel's Law applied, this is the redundancy you expect from collecting an entire sphere of diffraction data:
P1
P2, P21, C2
2
4
16
P3x, R3
12
P6x
12
P6x22
24
24
As a practical matter, some reflections that lie near the rotation axis are often "thrown away" due to large Lp corrections. The
Lorentz correction (L) is a correction for the amount of time that a reflection spends in diffraction condition. For reflections
lying near the rotation axis this correction may be very large and small variations in the estimation of this factor may introduce
large errors into the intensity estimate. For the same reason, you expect reflections that are furthest from the rotation axis to be
relatively weaker because they pass through diffraction condition (Ewald sphere) the fastest and spend the least time
diffracting. This also tends to be correlated with high resolution data, which tends to be weaker anyway.
The polarization correction corrects for the differential scattering of X-rays when the incident X-rays are polarized. The
form of the correction takes various forms, but (e.g.) for a circularly polarized beam the correction is: p = (1 + cos22)/2. At a
synchrotron the polarization is mostly in the plane of the synchrotron ring, which means that reflections whose diffraction
vectors are mostly perpendicular to this plane benefit the most from the polarization (intensities enhanced) - diffraction is
strongest in the directions perpendicular to the polarization plane. This is the reason that the oscillation axis at synchrotrons is
horizontal because those reflections that are passing the fastest through the Ewald sphere (therefore lower intensities recorded)
experience the most boost from the polarization effect. The Lp correction issue is also why it is often useful to have the
highest symmetry axis close to but not precisely aligned with the rotation axis in order to capture this "blind region" data via
symmetry relationships (if it is perfectly aligned then reflections related by this axis are still in the blind region).
Mosaicity
Crystals are not monolithic, they are composed of smaller fragments called mosaic blocks. These blocks are not perfectly
aligned with one another. Therefore a crystal has a mosaic spread that reflects the degree of orientational divergence of these
mosaic blocks. Good crystals have mosaic spreads of 0.2 degrees or less. Bad crystals have mosaic spread so 1.0 degrees or
more. Note that high mosaic spreads are often caused by less-than-optimal crystal cryo conditions, where the act of freezing
can move the mosaic blocks around with respect to each other. Hen Egg Lysozyme's tetragonal crystal form shows low
mosaicity (~0.1 degrees) at room temperature but closer to 1.0 degrees when frozen although this is a relatively unusual case
and many crystals fare better than this. In some situtations crystal annealing involving re-freezing the crystal can
substantially improve the crystal mosaicity properties. At other times selecting a smaller crystal or optimizing the composition
of the cryo buffer may help.
The estimate of mosaicity is often convoluted with the intrinsic beam divergence (the angular discrepancy from "perfectly
parallel"). So on a home source, with a more divergent beam, one is lucky to get less than 0.3 degrees for net crystal
mosaicity. On a synchrotron beamline with a nearly parallel beam I have seen as low as 0.12 degrees from a frozen crystal.
But I've also seen something close to 2.5 degrees on very ill-behaved crystals.
Note that it is perfectly possible to collect data on crystals with moderately high mosaicity as long as you take into
account the fact that overlaps are more likely (reflections persist over a larger angular range, so frames get more crowded). I
recommend that frame sizes should be at least the same size as the crystal mosaicity to avoid splitting up the reflections over
too many frames (although something like 2/3 of the mosaicity is the real lower limit). Empirically higher mosaicities tend to
be associated with crystal damage during handling or intrinsically poor crystal order.
Mosaicity is sometimes anisotropic, which can cause problems during data collection although you can refine a per-frame
mosaicity in Denzo and Scalepack. Often times, however, this just seems to model undesirable behavior like spot blurring due
to anisotropic crystal disorder, and its not always desirable to let the mosaicity fluctuate in an uncontrolled way during data
processing.
Wavelength
The home source with a copper anode is fixed at 1.54148 , but most synchrotrons have variable wavelengths (notable
exceptions are CHESS A1 and F1). The choice of wavelength depends on several factors, and if you are doing MAD the
absorption edge is by far the most dominant one. However for native data the decision is less obvious: Short wavelength
pros:
Less absorption (absorption varies as 3) means less absorption errors and background scatter
Smaller blind region (Ewald sphere has larger radius)
More compact diffraction pattern makes it easier to collect high resolution data
Short wavelength cons:
Less absorption means weaker diffraction and also possibly detector efficiency
Beam is often weaker (X-25's peak intensity is 1.1 )
If you are screening heavy atom data for substitution (e.g. Hg, Pt, Au crystals for potential MAD experiments) you can set the
wavelength to be around 1.0 which is the high or ultra-high energy remote for these edges and thus may contain some
anomalous signal. Setting the wavelength to 1.2 will be below the edge for these common derivative elements and you will
not get any anomalous signal.
Reducing Noise
Any pixel on the detector accumulates noise from a variety of sources:
X-ray scattering from your loop and meniscus
Air scattering from the path of the exposed direct beam in air
Zingers from radioactive decay of the Thorium in the CCD optical tapers
Electronic noise from detector circuitry (aka "dark current")
Cosmic rays
Cosmic rays tend to be a low contributor to the overall noise, but is the reason that image plates are erased just before use.
Image plates have low intrinsic electronic noise. CCDs have much higher noise but are cooled to minimize it. The "dark
current" images that the CCDs take before data collection are an attempt to correct for that electronic noise background.
Zingers tend to be relatively infrequent but you can see them on some CCD images - they thankfully tend to affect relatively
few pixels. The presence of zingers limits the amount of exposure time a CCD can be used in single exposure mode to about
90-120 seconds and beyond this point you have to collect pairs of images for each oscillation range to factor these effects out.
Non-diffraction X-ray scattering is however a big source of noise. The vast majority of the direct beam passes right through
the crystal and some of it is scattered by air or the crystal support (loop etc). You can tell this is a big effect because this is
why you see the beamstop shadow - the scatter is causing the shadow. You can reduce the air scatter component by reducing
the path of the direct beam in air and mostly this means moving your beamstop closer to your crystal. However the beamstop
does cast a shadow and you want to make sure you are able to collect your low resolution reflections too. (Potentially you can
move the beamstop in during the high resolution pass and back out during the low resolution pass). Air scatter falls off with
the square of the distance but the diffracted beams only fall off slowly with distance (air absorption and a slow spreading of
the spots due to mosaicity and beam divergence). Therefore you can reduce the air scatter by moving the detector further back
although you need to put it close enough to collect the highest resolution that you require. Air scatter also is reduced with
shorter wavelength.
For the same reason that reducing the amount of air the direct beam passes through is a good way to reduce background,
making sure that your cryo meniscus is not just one huge blob but resembles a thin film is also going to help. Theoretically
using 10 m rather than 20 m loops might help but I've found that 10 m loops end to move around a little in the cryo
stream.
Increasing Signal
The best way to increase the diffraction signal is to GROW A LARGER CRYSTAL. This is actually the only "free" way
to increase your signal. Other ways include:
Increasing exposure time (con: increases radiation damage)
Increase the number of diffraction images i.e. redundancy (con: increase radiation damage)
Increase wavelength (con: increases air scatter)
Increase the strength of the beam (e.g. move to the best wavelength for the optics, bigger collimator)
Shoot the crystal at multiple discrete locations and collect more data.
Anything that increases the number of photons that hit the crystal will increase the radiation damage. It's not obvious what the
trade off is between (e.g.) doubling the exposure time and doubling the number of images - both increase the signal/noise of
the final merged reflection. The former does so by increasing the signal/noise of each observed reflection and the latter does by
increasing the number of independent measurements for each unique reflection. Increasing the exposure time also increases
the background noise by the same factor, so it depends which statistical issue is dominant (counting stats where the doubling
the counts increases the signal/noise by 1.4; or variation in the background noise). Increasing beam brightness is exactly the
detectors (data is being collected on one while the other is being scanned). At 30 minutes per 1 degree frame a 70-degree data
collection will take 1.5 days. This is about the minimum number of frames required in point group 222 (orthorhombic).
However if you get your data collection strategy wrong you might need as much as 135 degrees, which would take about 3
days.
Although the start point for data collection doesn't affect the overall quality of the data (your crystal is effectively immortal,
you can always collect more data) it does radically affect your efficiency at screening multiple crystals. In summer, icing of
the sample may make 3 day data collections a difficult proposition - it pays to get complete data as fast as practical, and then
add more data as desired once the dataset is complete.
On home sources crystals nearly always show reduced maximum resolution compared to the same crystal on a synchrotron
beam line. There are two reasons for this:
the signal is lower
the noise is proportionally higher
Large crystals that diffract strongly to a well-defined upper limit of resolution probably won't show much difference between
home sources and synchrotrons, but these tend to be in the considerable minority.
X29 is of the order of 1000x brighter than the home source. Using these numbers, a 4 second exposure at X29 is equivalent to
a 71 minute exposure at home. In fact, the data you get at X29 is still better than that. At home we use 1.54 X-rays, and at
X29 you typically use 1.1 X-rays. At home the beam from the Yale optics is 0.3mm wide, and at X29 it is closer to 0.11mm
wide - about a 7.4x difference in cross-sectional area. For an 0.1mm (100 micron) crystal 80% of the beam is missing the
crystal at home, whereas only 20% is missing it at a synchrotron. This makes the difference in effective brightness even
greater (i.e. brilliance: photons/sec/area versus brightness: photons/sec). For large crystals this effect is smaller, obviously.
However at X29 the 1.1 X-rays actually interact with your protein crystal less strongly than the home source 1.54 X-rays.
On average this turns out to be a good thing (surprisingly) because the X-rays also interact with air less strongly too. It's been
known for a long while that the air-scatter by the direct beam is a major source of background noise (this is, after all, the
cause of the beamstop shadow). Absorption varies as 3 so 1.54 X-rays are scattered by air about 2.7x greater than by 1.1 .
So the weaker signal due to the weaker home source is combined with the proportionally higher background noise. Scatter
also happens from things like the loop material and the film of cryo that keeps your crystal in place, but the same principles
apply to these effects too. Higher background scatter leads to lower signal/noise since the variance in the background gives
rise to more inherent noise in the image. Some people have used helium-filled cones or bellows with mylar windows to reduce
air scatter (air scatters more strongly from diatomics like N2 than from monatomics like He).
There are other pro-synchrotron issues: spectral purity (the proportion of X-rays that are actually the wavelength we want) is
much higher at synchrotrons; beam divergence ("spread") is much less at synchrotrons so the spots are tighter on the detector
thus reducing the per-pixel noise component. A combination of all the factors (beam strength, air scatter, beam size, spectral
purity, beam divergence) heavily favors beamlines like X29 over home sources. For small crystals the situation can be
particularly dramatic with diffraction barely visible at home and collectible to high resolution at X29.
right. However much time is lost at synchrotrons from lack of preparedness and at the end of the day this corresponds to lost
data.
2
7
12
17
22
27
1.0227
1.0350
1.0509
1.0794
1.1115
1.1285
3
8
13
18
23
28
1.0248
1.0376
1.0490
1.0897
1.1075
1.1307
4
9
14
19
24
29
1.0143
1.0312
1.0679
1.0942
1.1207
1.1573
5
10
15
20
25
30
1.0319
1.0529
1.0657
1.0784
1.1348
1.1410
If these vary too much between frames use a "SCALE RESTRAIN 0.02" line within the Scalepack command file but make
sure you define the breaks between beam fills to avoid falsely restraining factors across legitimate discontinuities (e.g. here):
216
221
1.5645
1.5583
217
222
1.5583
1.5978
218
223
1.5526
1.5595
219
224
1.5594
1.4846
220
301
1.5781
0.1486
302
307
0.1490
0.1573
303
308
0.1512
0.1596
304
309
0.1555
0.1570
305
310
0.1567
0.1601
306
311
0.1572
0.1604
Similarly B-factors should vary only smoothly and you might want to use "B RESTRAIN 0.1":
New B factor
1
-0.94
6
-0.77
11
-0.84
16
-0.56
2
7
12
17
-0.73
-0.74
-0.75
-0.59
3
8
13
18
-0.79
-0.75
-0.82
-0.58
4
9
14
19
-0.83
-0.84
-0.63
-0.48
5
10
15
20
-0.74
-0.68
-0.66
-0.65
Notice in this case frame #1 does not have k=1 and B=0 but scaling works anyway. I probably forgot to include the
"REFERENCE FILM 1" line in the command file.
Scalepack normally post-refines the unit cell dimensions and detector geometry based on the entire dataset, which usually
results in more accurate cell parameters and addition of partial reflections across frame boundaries. We usually refine the cell
dimensions CRYSTAL and the mis-setting angles BATCH. You should not see much variation in crysx/y/z unless the crystal
is slipping (bad) or the integration has not gone smoothly. Always compare the post-refined mosaicity with the one you used
for integration and if necessary re-integrate the data if the values differ by more than 10%.
Film #
1
2
3
a
34.859
34.859
34.859
b
63.271
63.271
63.271
c
76.360
76.360
76.360
alpha
90.000
90.000
90.000
beta
90.000
90.000
90.000
gamma
90.000
90.000
90.000
crysz
-94.169
-94.164
-94.162
crysy
17.507
17.508
17.507
crysx mosaicity
-12.668
0.315
-12.668
0.315
-12.665
0.315
Scalepack then prints a list of new rejections, which should get shorter and shorter as you run the script multiple times (the
scaling converges).
The next table re-iterates some of the information we've seen before (per-frame scale and B-factors) and also lists the number
of overflows (reflections whose pixels are saturated) and partials (reflections that lie on more than one adjacent frame) and
fulls (all in one frame). In the case below we have quite a few overflows on each frame because this was the high resolution
pass from a good diffractor. This particular data collection also includes a low resolution pass to add those data back in.
1
2
3
4
5
6
7
8
IP
IP
IP
IP
IP
count
count
count
count
count
count
count
count
fitted,
fitted,
fitted,
fitted,
fitted,
of
of
of
of
of
of
of
of
no
no
no
no
no
1
2
3
4
5
1.0077
1.0227
1.0248
1.0143
1.0319
-0.94
-0.73
-0.79
-0.83
-0.74
1
1
1
0
0
0
2
0
0
2
6
3
3
53
41
37
34
43
4
0
0
0
0
1
5
0
0
0
0
0
6
7
0 1125
1 582
1 590
1 604
2 565
The next table is a very useful table that you can use to assess data collection strategy:
Summary of reflection intensities and R-factors by batch number
All data
Linear
Batch
# obs # obs > 1
<I/sigma> N. Chi**2
R-fac
1
945
945
13.5
0.993
0.037
2
1244
1243
13.4
1.050
0.039
3
1200
1195
12.7
0.936
0.037
4
1214
1213
13.4
0.880
0.035
5
1200
1196
12.8
0.891
0.039
6
1236
1235
13.1
0.895
0.037
7
1242
1239
13.7
0.972
0.037
8
1247
1240
13.4
0.870
0.037
9
1209
1209
13.7
0.920
0.036
10
1233
1232
13.2
0.938
0.039
11
1241
1236
13.5
0.825
0.035
12
1225
1218
13.7
0.826
0.037
13
1211
1207
13.4
0.803
0.034
8
699
731
701
690
697
There is a per-frame linear R-factor (% deviation between symmetry related reflections), an I/I (greater than 10 is a good
number, less than 5 indicates weak data) and a Chi**2. Denzo and Scalepack make a big deal about the error model generally speaking if your Chi**2 are much less than 1.0 you should decrease the error scale factor until the overall Chi**2
gets much closer to one, and if Chi**2 is much greater than 1.0 you increase the error scale factor. If you are using a typical
Scalepack command file the value of the error scale factor is a pretty good indicator of the quality of your data (1.0 - excellent
data, 1.5 is good data, 2.5 is bad data with a lot of systematic errors). Error scale factors for good data are 1.2-1.5 at
synchrotrons and 1.5-2.0 on home sources. Chi**2 is basically a measure of how well your estimated variances match the
observed variances within the data based on Rsymm.
The difference between #obs and #obs >1 will tell you if you are adding new data (adding completeness) or just adding more
of the same data (adding redundancy). Until your data is complete you should watch the difference between these two
columns to make sure you are not pointlessly collecting the same data over again. Only after your dataset is mostly complete
should you be looking to add more redundancy to it.
Last come a set of tables that summarise redundancy:
Shell
Lower Upper
limit limit
50.00 3.02
3.02 2.39
2.39 2.09
2.09 1.90
1.90 1.76
1.76 1.66
1.66 1.58
1.58 1.51
1.51 1.45
1.45 1.40
All hkl
>19
0
0
0
0
0
0
0
0
0
0
0
total
3472
3465
3432
3403
3376
3375
3364
3348
3278
2982
33495
>19
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
total
95.2
99.6
99.9
99.9
99.9
99.9
99.8
99.6
97.8
88.7
98.0
completeness:
Shell
Lower Upper
limit limit
50.00 3.02
3.02 2.39
2.39 2.09
2.09 1.90
1.90 1.76
1.76 1.66
1.66 1.58
1.58 1.51
1.51 1.45
1.45 1.40
All hkl
Average
I
66849.0
21930.9
12502.5
6243.9
3052.8
1728.3
1192.2
872.4
551.3
373.9
11889.5
Average
Norm. Linear Square
error
stat. Chi**2 R-fac R-fac
1409.8
962.8 0.964 0.026 0.028
482.0
338.0 1.675 0.040 0.046
304.1
232.2 1.304 0.040 0.044
190.5
159.9 1.057 0.048 0.049
127.1
115.5 0.904 0.061 0.061
102.0
96.8 0.814 0.084 0.081
93.9
90.8 0.797 0.110 0.101
90.8
88.9 0.752 0.141 0.125
94.6
93.8 0.694 0.194 0.173
106.5
106.0 0.667 0.247 0.216
306.9
232.8 0.981 0.036 0.031
This particular dataset goes to 1.4 but notice I have applied the usual criteria of cutting at the shell where the Rsymm reaches
the 20% range and the I/I drops to ~3.
It's evident that my previous criteria were far too conservative to the point of excluding reflection data that were useful in
refinement. Some people now advocate a <I>/<I> of 2.0 as a useful cutoff, but some go as low as 1.0. The Rsymm of the data
with a 2.0 cutoff is going to be in the 50% range, depending on redundancy. This might go against your intuition, but my own
experiments have indicated that the accuracy of the mean of this data (the <I>) is far more accurate than the Rsymm might
suggest - R-free and R-work values in the 25-35% range are not unprecedented in the outermost shells of data cut with a
<I>/<I> = 2.0 cutoff.
Here's a recently-published example of this, from a high resolution structure of the Rhomboid protease (Vinothkumar et al.
2010, EMBO J, advance online 1st October 2010):
GlpG native Acyl enzyme
Data Collection:
Resolution ()
(outer shell)
Rmerge
55.2-1.65
44.62-2.09
(1.74-1.65) 2.20-2.09
0.055 (0.575) 0.054 (0.394)
I/I
12.4 (2.4)
16.3 (2.9)
Completeness (%) 99.8 (100)
97.0 (85.4)
Redundancy
4.5 (4.2)
4.9 (3.5)
Refinement:
Resolution ()
34.77-1.65 31.16-2.09
(outer shell)
(1.74-1.65) (2.22-2.09)
Rwork/Rfree
0.192/0.218 0.198/0.242
(outer shell)
(0.26/0.275) (0.248/0.276)
Certainly it's hard to argue with outer shell R-free values of 27% on data cut at 2.4 and 2.9 respectively. The second dataset
shows signs of being cut more conservatively because the detector was too far out - notice the drop off in redundancy and
completeness in the outermost shells - or it could have been an anisotropy problem. Either way it's now clear to me that
cutting at 2.0 or even less is far more appropriate than cutting at 3.0 - there's lots of useful data out beyond 3 that we
used to discard.
structures. Even at 4.0 you can do little except describe the overall fold and most of the side-chains will not be resolved. At
3.5 you can start to describe the positions of side-chains, although such descriptions will be somewhat approximate. By 3.0
and beyond you can start to say something about specific interactions and relate them to biological function.
The minimum resolution for MIR and MAD structural solutions is around 4.5 , because any lower than that and the maps
become extremely difficult to interpret. This is also around the minimum useful resolution for molecular replacement. As in
everything, there are exceptions (e.g. many copies to average across), but not all that many. MIR and MAD require you to
actually measure good data, and not just "see a spot" at 4.5 .
Consequently, if your crystal diffracts to no better than 5.0 resolution under the best circumstances (e.g. radiation-damagelimited native data collection on your best highly-optimized crystal) then it's best to throw it in the trash can and save your
energy for something better (a new construct or sacrificing to a new god, etc).